Non-human genetically modified mammal lacking the alpha-fetoprotein

ABSTRACT

The present invention is related to a non-human genetically modified mammal comprising a mutation, a partial or total deletion of the genetic sequence encoding the wild type mammal alpha-fetoprotein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Phase under 35 U.S.C. § 371 of International Application No. PCT/BE00/00081, filed Jul. 11, 2000, which claims priority to U.S. Provisional Application No. 60/143,269, filed Jul. 12, 1999.

FIELD OF THE INVENTION

The present invention is related to a non-human genetically modified mammal, preferably a knock-out mouse, comprising a partial or total deletion of a genetic sequence encoding the alpha-fetoprotein (AFP) and used as a model for the study of fertilization or contraceptive methods and drugs.

The present invention is related to a non-human mammalian pluripotential embryonic stem cell comprising a partial or total deletion of a genetic sequence encoding a mammal alpha-fetoprotein (AFP).

The present invention is also related to study, testing and/or screening method and device of known or unknown compounds able to bind the mammal alpha-fetoprotein (AFP) and that may be used as agonist or antagonist of oestrogens to the mammal alpha-fetoprotein.

BACKGROUND OF THE INVENTION

Alpha-fetoprotein (AFP) is a glycoprotein present in the serum and a classical oncofetal marker. This protein is expressed at high levels during fetal life in the liver and the visceral endoderm of the yolk sac, and at lower levels in the developing gastrointestinal tract (Andrews et al., 1982; Tilghman and Belayew 1982), in the adult serum only trace amount are detected (Tilghman and Belayew 1982). The protein expressed by the embryos is secreted and present in the maternal blood circulation during gestation, the level of AFP concentration in the maternal serum is use to detect fetuses with spina bifida or Down's syndrome. The reason for this altered AFP level associated with those pathologies are not understood, but they have been used extensively in prenatal screening. The synthesis of AFP decreases dramatically after birth and only trace amounts are detected in adult liver. However expression of afp is associated with hepatocarcinomas and liver regeneration induced by partial hepatectomy or acute tetrachloride (CC14) intoxication. The control of afp gene expression has thus attracted much attention and it has been shown that afp expression is regulated by transcriptional mechanisms involving a large promoter and three distant enhancers (review of Chen et al. (1997)). Because AFP is synthesized during the G1 and S phases, it has been hypothesized that intracellular AFP affects cell growth (Leffert and Sell, 1974; Sell et al., 1975; Tsukada and Hirai, 1975; Belanger et al., 1978). The observation that AFP is able to bind estrogen led to the suggestion that AFP plays a role in the control of cell metabolism. In addition to binding estrogen, AFP, like albumin to which it is evolutionary related, is able to bind other steroids and endogenous and exogenous substances such as fatty acids, billirubin and various pharmaceutical agents suggesting that AFP may play a general transportation function. For the fetus, in this respect, AFP could serve as a modulator/modifier of various cell growth regulatory pathways during embryonic and fetal development in vertebrates by interacting and/or binding cytoplasmic chaperone proteins that normally escort nuclear receptors or transcription co-factors through the cytoplasm towards organelle interfaces (Mizejewski, 1995, 1985). AFP has also been proposed to protect the embryo against the maternal immune system, on the basis of the observation that addition of purified AFP into the culture of splenic or lymphnode mononuclear cells exerts a suppressive effect on antibody synthesis.

The different hypotheses proposed for AFP function(s) can be focused on the fetal life (stage at which the gene is strongly transcripted) since the protein is described as a fetoprotein.

At the present time, no document of the state of the art has suggested that the alpha-fetoprotein may play an essential role for female reproduction and fertility.

Aims of the Invention

The present invention aim to provide new models (animal models) as well as new methods and devices for the study, the testing and/or the screening of fertility or contraception methods, compounds and compositions intended for adult mammals (including humans) and/or for the study, the testing and/or the screening of new methods, compounds or compositions intended for the treatment and/or the prevention of osteoporosis.

SUMMARY OF THE INVENTION

The present invention is related to a non-human genetically modified mammal (preferably a knock-out mouse) comprising a mutation, a partial or total deletion in a genetic sequence encoding a mammal alpha-fetoprotein (AFP) described in GenBank (v00743).

Advantageously, said mammal comprises an heterozygous or homozygous mutation, partial or total deletion in the genetic sequence encoding a mammal AFP.

According to another embodiment of the present invention, said mammal is a sterile female comprising said homozygous mutation, partial or total deletion.

The present invention is also related to specific sequences such as primers that are used to identify if a mammal comprises said mutation, partial or total deletion heterozygously or homozygously.

Another aspect of the present invention is related to a non-human mammal pluripotential embryonic stem cell, preferably a mouse pluripotential embryonic stem cell comprising a partial or total deletion of a genetic sequence encoding a mammal AFP. Said stem cell can be advantageously used to obtain the non-human genetically modified mammal according to the invention by methods well known by the person skilled in the art described hereafter.

A further aspect of the present invention is related to the use of the non-human mammal according to the invention for the study, the testing and/or the screening of known or unknown anti-osteoporosis fertility and/or contraceptive methods, compounds or compositions.

A last aspect of the present invention is related to study, testing and/or screening methods and devices comprising the AFP or a portion of said AFP, preferably the AFP domain III comprising about 200 amino acids (as described by Festin (1999)), being fixed upon a solid support and used as a substrate for known or unknown compounds or compositions in a competitive test or method. Said device comprises also a medium comprising (possibly labeled) oestrogens. The device according to the invention can be a chromatographic column upon which the AFP or the portion thereof is fixed or a study, testing and/or screening kit comprising disposed separately the various media necessary for said study, testing and/or screening.

According to a preferred embodiment of the present invention, said device or kit may comprise a cell having integrated an oestrogen-sensitive (proloactine) promoter gene whose activation may result from the fixation of known or unknown compounds or compositions upon the AFP. Said known or unknown compound or composition could be used advantageously as an agonist of an oestrogen.

The present invention is also related to this unknown agonist or antagonist of oestrogens screened and identified by the method and the device according to the invention. This unknown molecule finds an application in the field of fertility and/or contraceptive methods and compositions and/or is used also for the treatment and/or the prevention of osteoporosis.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executed in color. Copies of this patent or application publication with colored drawings will be provided by the Office upon request and payment of the necessary fees. FIGS. 3 to 6 are executed in color.

FIG. 1A illustrates the targeted disruption of the afp gene to generate alleles which are deleted for most of the sequence of exon 1, for exon 2 and for exon 3.

FIG. 1B illustrates the detection of homologous insertion by Southern blot analysis.

FIG. 1C illustrates a Southern blot analysis of the progeny of homozygous (−/−) chimeric animals.

FIG. 2A is a Northern blot analysis of total RNA from embryonic liver.

FIG. 2B is a Western blot analysis of embryonic liver and amniotic fluid protein extracts.

FIGS. 3A to 3H illustrate expression of the lacZ gene in embryonic and adult tissue. FIGS. 3A, 3G and 3H illustrate expression in adult liver and gut cells.

FIGS. 3B to 3F illustrate expression of the lacZ gene in embryonic tissues.

FIG. 4A illustrates the structure of the ovary (arrow) and uterus of an adult afplacZ1/lacZ1(−/−) female. FIG. 4B illustrates the ovary from a 12 week old afplacZ1/lacZ1 animal. FIG. 4C illustrates the ovary from a 12 week old mutated afplacZ1/lacZ1 female. FIG. 4D illustrates the general histological structure of afplacZ1/lacZ ovaries. FIG. 4E illustrates the structure of wild type ovaries at 4 months.

FIG. 5A illustrates the nucleotide sequence of the afp gene. FIG. 5B illustrates the amino acid sequence of the afp protein.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Generation of Mice Carrying a Germ-Line Mutation in the Afp Gene

A clone containing a 129 genomic fragment of AFP loci was isolated from a lambda library. The library was screened with a probe containing the mouse afp promoter. The genomic insert of about 16 kb was subcloned into pKIL-PCR2 (Gabant et al., 1997). The targeting vector (pAFP K.O-1), consists of two recombination arms. The 5′ arms (2.5 kb) were generated by polymerase chain reaction (PCR) using the following primers: N-Mer1: agagcggccgcggaagtgacaaagcagaacc (SEQ ID NO: 1) annealing to the MerIsequence of the afp enhancer 1 (Godboute et al. (1988)) and a primer of the X-exon1: agactcgagggatgagggaagcgggtgtg (SEQ ID NO: 2) complementary to the afp exon1. The PCR fragment generated using Pfu polymerase (Stratagene) was cloned in the pCR-blunt vector (Invitrogen).

The 3′ arms were subcloned from the lambda into pBSIIKS+ vector (Stratagene). The 5′ recombination arm was introduced upstream the 3′ recombination arm. The IRES lacZ/neo reporter-selective cassette was introduced between these recombination arms. The tk2 negative selective marker was introduced into the SalI site to generate pAFP KO-1. This construction was linearized with NotI and electroporated into E14 ES cells. Correctly targeted clones were identified by Southern blot analysis using an external probe from the 5′ region.

ES Cell Injections and Animal Genotyping

Recombinant ES cells carrying the targeted allele were injected in C57BL/6J blastocysts. Animals were genotyped by extraction of DNA from tails.

RNA Isolation, Northern Blot Analysis

Total RNA was isolated using Trizol (Gibco BRL) extraction according to the manufacturer instructions. For the Northern analysis 20 μg of total RNA were electrophoresed and transferred to nylon membranes as described. Filters were then hybridized.

Western Blot Analysis

Proteins were separated by SDS-PAGE using 7.5% polyacrylamide gels in a Bio-Rad Mini Protean gel chamber and blotted onto Nitrocellulose filters in a Bio-Rad Trans Blot chamber according to the manufacturer's instructions. Proteins were detected using anti-AFP, anti-Albumin; anti Betagalactosidase serum (ICN Biochemicals) the signal was detected with ECL detection system (Amersham).

LacZ Reporter Gene Expression

To isolate embryonic stages, natural matings were set up and presence of a vaginal plug at noon the following day was taken as 0.5 days of gestation. Staged embryos were stained with X-Gal as wholemounts as described by Forrester et al. (1996). For cryostat sectioning, tissues were embedded in optimal cutting temperature (OTC) compounds (Miles, Inc., Elkart, Ind.), and sections stained for X-Gal were counterstained with haematoxylin and eosin, and mounted.

Targeted Mutagenesis of the afp Gene

The afp gene was disrupted by gene targeting in embryonic stem (ES) cells. The lacZ reporter was introduced in afp gene by homologous recombination and placed under the control of the AFP promoter-enhancer region. The resulting allele is deleted for most of the sequence of exon1, for exon2 and 3 (see FIG. 1A) and homologous insertion was detected by Southern analysis (see FIG. 1B). To test the functionality of the reporter one may take advantage of the observation that AFP is expressed in embryoid bodies (Abe et al., 1996). Reporter gene activity is highly turn on in some cells of these bodies (see FIG. 2A). No expression of the reporter was detected in undifferentiated ES cells grown in the presence of LIF.

ES cells afp lacZ1/+ were injected into C57BL/6J blastocysts. Chimeric animals were obtained and mated with outbred CD1 or inbred 129/CGR to test for germ line transmission. Phenotypically normal heterozygous mice afp lacZ1/+ were generated and detected by Southern blot (see FIG. 1C).

Reporter Expression Analysis

Expression of the lacZ reporter gene expression in embryonic and adult tissues was analyzed. As shown in FIG. 3 the β-galactosidase activity was detected in predicted embryonic tissues. In the visceral endoderm only patches of cells were observed to turn the reporter strongly on. In the adult tissues tested specific staining was only detected in cells of the liver and in cells of the gut.

Intercrosses of heterozygotes (afp lacZ1/+) gave rise to viable, apparently normal homozygous mutant mice at a Mendelian ratio in CD1 and C57/B16. On the other hand, a significant divergence was observed in the 129 background (Table 1). To determine whether the targeted allele indeed results in a null mutation, total RNA from the liver of embryos was analyzed by Northern blot hybridization (FIG. 2A). A strong signal at 2.2 kb corresponding to the afp transcript was detected in wild type and heterozygous embryos. No signal was detected in RNA samples extracted from homozygous embryonic liver, showing that the recombination disrupted the afp transcript in these animals. A Western blot was also performed on embryonic liver and amniotic fluid protein extracts (FIG. 2B) and a strong signal was detected with the wild type extracts while no band corresponding to the AFP was visible in the homozygous extracts demonstrating that these animals do not express AFP.

TABLE 1 Intercrosses Parents Offsprings Strains Male Female +/+ +/− −/− CD1 +/− +/− 108 258 102 129 +/− +/−  34  48  13 C57/black-6 +/− +/−  19  43  19 AFP is Required for Female Fertility

The Mendelian ratio obtained in CD1 and C57/black-6 background demonstrates that there is no reduction in the intercrosses. On the other hand, the divergence observed in 129 background suggests that AFP is involved in the gestation and that its importance is only revealed in some genetic contexts. In these litters derived from intercrossing heterozygous animals, homozygous embryos develop in the presence of their wild type and heterozygote littermates. AFP produced by these embryos is secreted and present in the maternal serum. To determine whether afp lacZ1/lacZ1 mice are able to develop in the complete absence of AFP afp lacZ1/lacZ1 males and afp lacZ1/lacZ1 females were mated. No pups were obtained from these intercrosses suggesting an essential role of AFP for development and/or fertility (see Table 2). To test if fertility was affected, afp lacZ1/lacZ1 males and females were mated with wild type animals. Males homozygous for an afp disrupted allele appeared fertile and sired offspring but homozygous females never produce any live offspring. To test if natural matings occur with the afp lacZ1/lacZ1 females leave with wild type males the vaginal plug were checked. No plugs were detected with those females, showing that the origin of the observed infertility is due to an absence of mating. To identify the defect underlying the reproductive capacity of homozygous females, the reproductive system of those animals was analyzed. The reproductive system of the afp lacZ1/lacZ1 females was dissected and at this stage of the analysis appeared complete A major anatomical difference is notable between ovaries from afp lacZ1/lacZ1 and afp +/+: the ovaries of afp lacZ1/lacZ1 are smooth, this observation suggests that those females do not ovulate. Histological analysis of mature afp lacZ1/lacZ1 ovaries shown that their homozygous tissues do not contain corpus lutea, the lack of these structure is indicative of the absence of ovulation (see FIG. 4). The afp lacZ1/lacZ1 ovaries contain follicles at the different stages of maturation, this suggests that the default of AFP during the development has no effect on the female gametogenesis. However, the presence of follicles does not prove that these gametes are competent for maturation. To test the competence of the afp lacZ1/lacZ1 follicles, they were dissected out and analyzed their potential of maturation in vitro. Complete maturation was obtained in vitro with the dissected oocytes from afp lacZ1/lacZ1 animals. Taken together these data indicates that those females do not ovulate properly and thus that a signal needed to trigger ovulation is absent in the afp lacZ1/lacZ1 mice.

TABLE 2 Phenotypical analysis Parents Offsprings Male Female +/+ +/− −/− −/− −/− (2)  0 0 0 −/− +/+ (6)  0 71 0 +/+ −/− (13) 0 0 0

Table 2: Infertility phenotype of the afp^(lacZ1/lacZ1) (−/−) homozygous animals were mated, no offsprings were obtained from these matings. To test fertility of the afp^(lacZ1/lacZ1) (−/−) males and females, homozygous males and females were mated with wild type (+/+) animals. For the different breedings the number of mating is given in brackets.

It was also observed that afp^(lacZ1/lacZ1) follicles are able to mature normally in vitro (data not shown) suggesting that the defect could be in a signal required to trigger ovulation and indeed ovulation can be induced in these animals by a superovulation protocol (Table 3).

TABLE 3 Ovulation induction in afp^(lacZ1/+) and afp^(lacZ1/lacZ1) females Mice injected Number of oocytes obtained afp^(lacZ1/+) females (9 weeks) 37 afp^(lacZ1/lacZ1) females (9 weeks) 31

Table 3: Induction of ovulation in afp^(lacZ1/lacX1) females. afp^(lacZ1/lacZ1) and afp^(lacZ1/+) females were hormonally treated to induce ovulation. The average number of postovulation oocytes obtained from 6 individual females tested.

Mouse Genotyping by PCR

In order to identify a possible mutation or deletion in the afp gene, a specific genotyping of afp −/− mice by PCR has been developed. Two primers are used for the first afp#1 anneals in the afp promoter region (−116 bp to −137 bp: according to the +1 of the mouse afp gene). The second primer afp#2 is complementary to the first exon of the mouse afp gene (+141 bp to +160 bp: according to the +1 of the mouse afp gene): sequence deleted in the afp −/− knock-out mouse described in the text.

Sequence afp#1: cccctgctctgttaattattg (SEQ ID NO: 3) Sequence afp#2: gaaaatagctcccaagtcac (SEQ ID NO: 4)

No amplification product (300 bp) was observed in afp −/− animals. This amplification is present in wild type and heterozygous mice.

To differentiate +/− from wild type, a second PCR is performed using two primers giving an amplification on the DNA introduced in the genome of the transgenic animals (this sequence is not present in wild type animals).

For the knock-out mouse for afp described in the text: a couple of primers complementary to lacZ (E. coli gene) can be used.

lacZ#1: acaacgtcgtgactgggaaaac (SEQ ID NO: 5) lacZ#2: taatgggataggttacgt (SEQ ID NO: 6)

Those primers will only give a signal (287 bp) on +/− and no signal on wild type DNA samples.

The physiological role of the alpha-fetoprotein, the most abundant serum protein expressed by mammalian embryos remains to be establish. This protein related to albumin excreted by the embryos into the maternal blood circulation has attracted attention and due to its abundance it has been postulated that the presence of this protein was essential for embryonic development. To determine the function of AFP this gene was disrupted by homologous recombination in embryonic stem cells. Surprisingly homozygous afp^(lacZ1/lacZ1) are viable showing that expression of AFP by the embryo itself is not require for normal and complete development. This phenotype shows that the structure of the homozygous females was generally maintained. The different stages of ovocytes maturation are founded in these ovaries and shown to accomplish their maturation in vitro. Ovulation was induced in these females by hormonal induction and ovocytes were produced. Although AFP is synthesized at a high level during fetal life (mainly by the liver and the visceral endoderm of the yolk sac), low level of AFP mRNA has been reported in different other fetal and adult tissues as well as in adult rats. However, the level of expression in such tissues is very low. Another explanation is that the ovulation in those females is affected by the lack of AFP during development.

The fact that in adults afp^(lacZ1/lacZ1) ovulation can be induced argues for the absence in those females of a signal needed to trigger ovulation. This suggests that AFP is involved in the transportation of an element needed for ovulation in the adults.

By the disruption of the mouse AFP, one may show that fetal serum protein is required for females ovulation.

The fact that analbuminic rats are fertile shows that at least albumin cannot rescue AFP. This show that albumin and AFP plays two different role and that AFP is involved in the function of females ovaries.

The specific phenotype of the non-human mammals according to the invention is also illustrated in the enclosed FIG. 4, which presents the anatomical and histological analysis for the afp^(lacZ1/lacZ1) ovaries:

-   A: Structure of the ovary (arrow) and uterus of an adult     afp^(lacZ1/lacZ1) (−/−) female; -   B: Ovary from 12 weeks old afp^(lacZ1/lacZ1); -   C: Ovary from a 12 weeks old wild type female: the surface     distortions are due to the presence of large type follicles, whereas     afp^(lacZ1/lacZ1) ovaries are smooth; -   D: The general histological structure of the afp^(lacZ1/lacZ1)     ovaries is not affected and mature Graafian follicles are present in     those tissues (section from a fourth month old female); -   E: At 4 months, the wild type ovaries exhibit large corpus lutea,     indicative of successful ovulation. Those structures are never found     in afp^(lacZ1/lacZ1) ovaries.

Therefore, the non-human female mammals that present homozygously said mutation, partial or total deletion in the afp gene do not cycle. The surface of the ovaries is smooth and is not characterized by the presence of large follicles. Their histological structure is not generally affected, and Graafian follicles are identified in the tissues but no large corpus lutea is present.

Furthermore, females comprising an homozygous mutation or partial or total deletion in the afp gene do not allow uterus nidification of an embryo.

Additional experiments have shown that the afp gene is not essential for the survival of the mice. Indeed, as females give birth to severa animals, it was possible that the afp^(−/+) may survive to brothers and sisters (afp^(+/+) or afp^(+/−)) simultaneously present in the uterus of the female.

Therefore, in order to extrapolate the observed phenotype and genotype to the human population, the inventors have shown that blastocysts implanted one by one in pseudogravite females will obtain the birth of alive afp^(−/−) animals that present the same phenotype as above-described (fertile males, sterile females).

Therefore, the afp^(−/−) phenotype corresponds to alive sterile females, which is a phenotype that may exist in the mouse population as well as in the human population. 

1. A sterile female genetically modified mouse which does not undergo menstrual cyclization, wherein the genome of said mouse comprises a mutation, a partial deletion or a total deletion in each allele of endogenous genetic sequence encoding the wild type alpha-fetoprotein (AFP), wherein said mutation, partial deletion or total deletion results in loss of expression of a functional AFP in both alleles.
 2. The sterile female genetically modified mouse of claim 1, wherein said sterile female genetically modified mouse is homozygous for a mutation, a partial deletion or a total deletion in endogenous genetic sequence encoding a functional alpha-fetoprotein (AFP).
 3. A method for identifying an agent for use in increasing fertility, comprising: obtaining a sterile female genetically modified mouse which does not undergo menstrual cyclization wherein the genome of said mouse comprises a mutation, a partial deletion or a total deletion in each allele of endogenous genetic sequence encoding the wild type alpha-fetoprotein (AFP), wherein said mutation, partial deletion or total deletion results in loss of expression of functional AFP in both alleles; contacting said sterile female genetically modified mouse with said agent; and determining the effects of said agent on fertility in said sterile female genetically modified mouse. 